Oligonucleotides that bind sequence specifically to complementary nucleic acids (i.e. sense strand) by hydrogen bonding so as to inhibit gene expression are commonly referred to as antisense oligonucleotides. These synthetic oligonucleotides bind to target (MRNA) and thereby inhibit translation of the messenger RNA. This antisense principle (Uhlmann, E. et al., Chem. Reviews, 1990, 90, 543-584; and Stein, C. A. et al., Cancer Res., 1988, 48, 2659-2688) is used in nature to regulate gene expression. This antisense principle has been used in the laboratory not only to inhibit but also to activate gene expression. Zamecnik and Stephenson were the first to propose, in 1978, the use of synthetic oligonucleotides for therapeutic purposes (Stephenson, M. L.; and Zamecnik, P. C., Proc. Natl. Acad. Sci. USA, 1978, 75, 280 and 285). The specific inhibition of antisense polynucleotide is based on the specific Watson-Crick base pairing between the heterocyclic bases of the antisense oligonucleotide and of viral nucleic acid. The process of binding of the oligonucleotides to a complementary nucleic acid is called hybridization. An oligomer having a base sequence complementary to that of an mRNA which encodes protein necessary for the progress of the disease is of particular interest. By hybridizing specifically to the mRNA, the synthesis of proteins encoded by the mRNA may be disturbed.
The preparation of unmodified oligonucleotides, i.e., oligonucleotides having a DNA structure, has been the center of interest for many research groups in the past decade. The synthesis via phosphoramidites according to Caruthers (McBride, L. J.; and Caruthers, M. H., Tetrahedron Letts., 1983, 24, 245), originally introduced by Letsinger (Letsinger, R. L.; and Lunsford, W. B., J. Amer. Chem. Soc., 1976, 98, 3655) as the phosphite triester method, is currently the most efficient method for the preparation phosphodiester oligonucleotides. When normal, i.e., unmodified, oligonucleotides are used as antisense oligonucleotides, the problems of instability to nucleases and insufficient membrane penetration have arisen. For antisense oligonucleotides to be able to inhibit translation they must reach the interior of the cell unaltered. The properties useful for oligonucleotides to be used for antisense inhibition include: (i) stability of the oligonucleotides towards extra- and intracellular enzymes; (ii) ability to penetrate through the cell membrane; and (iii) ability to hybridize the target DNA or RNA (Agarwal, K. L. et al., Nucleic Acids Res., 1979, 6, 3009; Agawal, S. et al., Proc Natl Acad Sci. USA. 1988, 85, 7079). Thus, it is of interest to provide polynucleotide analogs that have superior properties for use as antisense or for use as primers or hybridization, probes.
Modified polynucleotides have been synthesized in the past, these polynucleotide modifications include methylphophonates, phosphorothioates, various amidates and the sugar moieties of the nucleic acid species. These backbone substitutions confer enhanced stability to some degree but suffer from the drawback that they result in a chiral phosphorous in the linkage, thus leading to the formation of 2.sup.n diastereomers where n is the number of modified diester linkages in the oligomer. The presence of these multiple diastereomers considerably weakens the capability of the modified olgonucleotide to hybridize to target sequences. Some of these substitutions also retain the ability to support a negative charge and the presence of charged groups decreases the ability of the compounds to penetrate cell membranes. There are numerous other disadvantages associated with these modified linkages, depending on the precise nature of the linkage.
Some oligonucleotide analogs containing sugar modifications have been synthesized. Previously used sugar modifications of (deoxy)ribose nucleic acids include .alpha.-DNA, homo DNA, morpholino and thio nucleosides and Peptide Nucleic Acids (PNA) to provide what has been perceived to be improved structures, especially structures which have improved cell uptake. The general synthetic scheme for arriving at such analogs has been to involve the primary hydroxyl group of a nucleoside or its nucleotide, either bound to a polymeric carrier or to a sequence-specified 3'-nucleotide with phosphorus atom in either the pentavalent or trivalent oxidation state. Specific coupling procedures have been referred to as the phosphite triester (phosphoramidite), the phosphorus diester, and the hydrogen phosphonate procedures. Commercially available monomers and polymeric carriers-bound monomers are available for such methods having protected bases (G, A, C, T, U and other heterocycles) along with protected phosphorus atoms to allow storage and prevent non-specific reactions during the coupling process.
Nucleic acid species containing modified sugars, nonionic backbones or acyclic polyamides (PNA) having, to some degree, one or more of the following properties useful for gene modulation: to enhance the duplex stability (hybridization efficiency), increased target specificity, stability against nucleases, improved cellular uptake, and assistance in the important terminating events of nucleic acids (e.g. RNase H activity, catalytic cleavage, hybridization arrest, and others). It has also been suggested to use carbonate diesters. However, these compounds are highly unstable, and the carbonate diester link does not maintain a tetrahedral configuration exhibited by the phosphorous in the phosphodiester. Similarly, carbamate linkages, while achiral, confer trigonal symmetry and it has been shown that poly dT having this linkage does not hybridize strongly with poly dA (Coull, J. M. et al., Tetrahedron Letts., 1987, 28, 745; Stirchak, E. P. et al., J. Org. Chem., 1987, 52, 4202).
More recently, reports of acyclic sugar analogs have appeared in the literature (Augustyns, K. A. et al., Nucleic Acids Res., 1991, 19, 2587-2593). Incorporation of these acyclic nucleosides into oligonucleotides caused a drop in Tm, depending on the number of linkers built in the oligomers. These oligonucleotides are found to be enzymatically stable and form base pairing with the complementary sequence. Given the shortcomings of polynucleotides and known polynucleotide analogs, it is of interest to provide new polynucleotide analogs for use in antisense inhibition and other techniques employing oligomers.
Such attempts at modifying both the sugar and the backbone components have some shortcomings for use as therapeutics and in other methods. Hence, still greater improvements in these qualities is required before effective therapeutics, diagnostics and research tools become available. Accordingly, there is a long-felt need for improved oligomer analogs of oligonucleotides as pharmaceuticals compounds.
The present invention provides novel oligonucleotides, and structural precursor thereof, which have improved resistance to nuclease digestion, and which have increased stability under physiological conditions, and which can be neutral or positively charged that could enhance cell permeation. Furthermore, the novel oligonucleotides of the present invention improved hybridization properties with respect to nucleic acid hybridization targets.
The oligomers of the present invention are generally characterized as comprising a series of constrained linkers or monomers that is appropriate for binding of heterocyclic bases to a target nucleic acid in a sequence specific manner. The constrained linkers described herein, when incorporated into oligomers, may have a force greater than a single hydrogen bond, thereby favoring formation of the binding competent conformation.
The nucleomonomers of the present invention are generally characterized as moieties or residues that replace the furanose ring, that is found in naturally occurring nucleotides, with an amino acid or a modified amino alcohol residues. Exemplary monomers and oligomers of the invention are shown in formulae 1 through 41. Incorporation of these monomers described herein into oligonucleotides permits synthesis of compounds with improved properties, these improved properties include (i) increased lipophilicity which results from eliminating the charge associated with phosphodiester linkages (Dalge, J. M. et al., Nucleic Acids Res., 1991, 19, 1805) and (ii) resistance to degradation by enzymes such as nucleases. Oligomers containing these monomers may be quite stable for hybridization to target sequences and superior to unmodified nucleoside residues in one or more applications.